Tfiis is a K99 to ROO transition application which seeks further support for studying the association of four opioid receptor genes (0PRM1, 0PRD1, 0PRK1, and 0PRL1) and four opioid peptide genes (POMC, PDYN, PENK, and PNOC) genes and substance dependence (SD) in African Americans (AAs) and European Americans (EAs). In the K99 phase, we identified several opold receptor and peptide gene variants which were significantly associated with SD. However, since only a limited number of variants were selected and tested for their association with SD, the causal variant might have been ignored. In addition, it is unknown whether the identified SD-associated variants are functional or they are just in linkage disequilibrium with the causal variant. Therefore, in the ROO phase, we need to further address these Issues. We propose to (1) analyze the association of both common and rare variants with SD by resequencing the opioid receptor and peptide genes in our cases and controls using the next-generation sequencing technique;(2) examine the function of SD-assoclated opioid receptor and peptide gene variants using several approaches including receptor binding assays, Western blotting, real-time quantitative polymerase chain reaction, allelic expression Imbalance assay, electrophoretic mobility gel shift assay, and liclferase reporter gene assay;and (3) Investigate whether DNA metylatlon levels In the regulatory regions ofthe opioid receptor and peptide genes are significantly different between SD affected cases and healthy controls. The proposed study will improve our understanding about the Influence of opioid receptor and peptide gene variants on SD and determine whether epigenetic modification ofthese genes can Increase vulnerability to SD. In addition, it will generate sufficient data for a future ROI project aimed at (1) establishing a set of opiod receptor and peptide gene markers as predictors of SD;and (2) investigating the contribution of variants In these genes to the outcome of SD treatment.